突变和修饰IFN-α2b基因的5’和3’端对其在E.coli表达的调控作用

Mutagenesis and modifications of IFN -α2b gene affect the expression level in E.coli

目的对人 IFN- α2 b基因进行突变和修饰 ,从翻译水平上调控 IFN- α2 b基因在大肠杆菌中的表达。方法采用PCR技术 ,人工合成寡核苷酸引物 ,对人 IFN- α2 b基因进行了改造 :在不改变氨基酸的前提下 ,将 5’端编码区的四个 G、C位点突变为 A、T;去除 3’端非编码区。将突变和修饰后的基因片段分别克隆入原核表达载体 p BV32 1,在大肠杆菌中进行表达。对表达产物进行活性效价测定。结果 3’端删除非编码区 ,表达水平比删除前提高 4倍 ;5’端四个位点突变 ,表达水平比突变前降低 2倍。结论 IFN-α2 b基因的 3’端非编码区抑制其在原核系统的表达 。

ObjectiveTo investigate the effects of mutation and modification of human IFN α2b gene and translational control of the expression of IFN α2b gene in E.coli .MethodsPCR was pertormed as follows:four G and C bases in 5’ terminal were substituted with A or T,without altering the amino acid sequence;the downstream of stop codon TAG in the 3’terminal non coding region was removed.The mutated and modified genes were then cloned into expressing vector pBV321 and expressed in E.coli respectively. The activity and potency of expressed proteins were determined.ResultsThe highest level of expression was obtained by removal of 3’ terminal non coding region(four times as much as the original value);by mutation of 4 sites the 5’ terminal,the expression level was 0 5 times as compared to that of the original level.ConclusionIFN α2b gene 3’terminal non coding region hinders the expression in E.coli ,and it is feasible to increase the expression level by removal of the region.